Antibodies that specifically bind receptor

ABSTRACT

The cDNA that encodes a glycoprotein receptor from the tobacco hornworm which binds a  Bacillus thuringiensis  toxin has been obtained and sequenced. The availability of this cDNA permits the retrieval of DNAs encoding homologous receptors in other insects and organisms as well as the design of assays for the cytotoxicity and binding affinity of potential pesticides and the development of methods to manipulate natural and/or introduced homologous receptors and, thus, to destroy target cells, tissues and/or organisms.

RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of U.S. Ser. No08/326,117, filed Oct. 19, 1994.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] Work resulting in the present invention was supported in part byResearch Agreement 58-319R-3-011 from the Office of InternationalCooperation and Development, U.S.D.A. and by Cooperative Agreement58-5410-1-135 from the Arthropod-Borne Animal Disease Laboratory,Agricultural Research Service, U.S.D.A. and by Grant HD-18702 from theNational Institutes of Health. The U.S. government has certain rights inthis invention.

TECHNICAL FIELD

[0003] The invention relates to receptors that bind toxins from Bacillusthuringiensis and thus to pesticides and pest resistance. Moreparticularly, the invention concerns recombinantly produced receptorsthat bind BT toxin and to their use in assays for improved pesticides,as well as in mediation of cell and tissue destruction, dissociation,dispersion, cell-to-cell association, and changes in morphology.

BACKGROUND ART

[0004] It has long been recognized that the bacterium Bacillusthuringiensis (BT) produces bactericidal proteins that are toxic to alimited range of insects, mostly in the orders Lepidoptera, Coleopteraand Diptera. Advantage has been taken of these toxins in controllingpests, mostly by applying bacteria to plants or transforming plantsthemselves so that they generate the toxins by virtue of theirtransgenic character. The toxins themselves are glycoprotein products ofthe cry gene as described by Höfte, H. et al. Microbiol Rev (1989)53:242. It has been established that the toxins function in the brushborder of the insect midgut epithelial cells as described by Gill, S. S.et al. Annu Rev Entomol (1992) 37:615. Specific binding of BT toxins tomidgut brush border membrane vesicles has been reported by Hofmann, C.et al. Proc Natl Acad Sci USA (1988) 85:7844; Van Rie, J. et al. Eur JBiochem (1989) 186:239; and Van Rie, J. et al. Appl Environ Microbiol(1990) 56:1378.

[0005] Presumably, the toxins generated by BT exert their effects bysome kind of interaction with receptors in the midgut. The purificationof a particular receptor from Manduca sexta was reported by the presentinventors in an article by Vadlamudi, R. K. et al. J Biol Chem (1993)268:12334. In this report, the receptor protein was isolated byimmunoprecipitating toxin-binding protein complexes with toxin-specificantisera and separating the complexes by SDS-PAGE followed byelectroelution. However, to date, there has been no structuralinformation concerning any insect receptor which binds BT toxin, norhave, to applicants' knowledge, any genes encoding these receptors beenrecovered.

DISCLOSURE OF THE INVENTION

[0006] The present invention is based, in part, on the isolation adcharacterization of a receptor that is bound by members of the BT-toxinfamily of insecticidal proteins, hereinafter the BT-R₁ protein. Thepresent invention is further based on the isolation and characterizationof a nucleic acid molecule that encodes the BT-toxin receptor,hereinafter BT-R₁ gene. Based on these observations, the presentinvention provides compositions and methods for use in identifyingagents that bind to the BT-R₁ protein as a means for identifyinginsecticidal agent and for identifying other members of the BT-R₁ familyof proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 show the nucleotide sequence and deduced amino acidsequence of cDNA encoding the BT-R₁ protein from M sexta.

[0008]FIG. 2 (panels a and b) shows a comparison of amino acid sequencesof cadherin motifs (BTRcad-1 to 11) in BT-R₁ to those of othercadherins.

[0009]FIG. 3 shows a block diagram of the cadherin-like structure ofBT-R₁.

[0010]FIG. 4 shows the clone characterization of the BamHI-SacI fragmentof BT-R₁. LM is HindIII cut Lambda marker; UP is the uncut plasmidclone; NP is NsiI cut plasmid; XP is XhoI cut plasmid; BSP is BamHI andSacI cut plasmid showing the cloned fragment from BT-R₁; RM is mRNA sizemarker; and RT1 and RT2 are transcribed mRNAs from the cloned BT-R₁fragment.

[0011]FIG. 5 illustrates the detection of protein expression from theplasmid containing the Bam-Sac fragment of BT-R₁ using ³⁵S-methionine asa tag. LCR is a luciferase control mRNA to show that the rabbitreticulocyte lysates are functional; RR1 and RR2 are expression productsof the Bam-Sac fragment of BT-R₁ produced in rabbit reticulocytes frommRNA; LCT is a luciferase control plasmid to show that thetranscription/translation kit is functional; and TT1 and TT2 areexpression products of the Bam-Sac fragment of BT-R₁ produced in atranscription/translation kit.

[0012]FIG. 6 shows a radio-blot of the Bam-Sac fragment of BT-R₁ with¹²⁵I-labeled Cry1Ab. BBMV is the brush border membrane vesicles from themidgut of M. sexta containing the wild-type BT-R₁ receptor protein; RBKis a rabbit reticulocyte blank; RR1 and RR2 are the expression productsof the Bam-Sac fragment of BT-R₁ produced in rabbit reticulocytes frommRNA; TBK is a transcription/translation kit blank; TT1 and TT2 areexpression products of the Bam-Sac fragment of BT-R₁ produced in atranscription/translation kit. The arrows point to two of the bands.

[0013]FIG. 7 shows the presence of a BT-R₁ homologue in Pink Bollwormand European Corn Borer identified using toxin binding similar to thatused to identify the original BT-R₁ clone.

[0014]FIG. 8 shows the binding of Cry1Ab to fragments of the BT-R₁protein.

MODES OF CARRYING OUT THE INVENTION

[0015] I. General Description

[0016] The present invention is based, in part, on the isolation andcharacterization of a novel protein expressed in the midgut of Manducasexta that binds to members of the BT-toxin family of proteins,hereinafter the BT-R₁ protein. The present invention specificallyprovides purified BT-R₁ the amino acid sequence of BT-R₁ as well asnucleotide sequences that encode BT-R₁. The BT-R₁ protein and nucleicacid molecules can serve as targets in identifying insecticidal agents.

[0017] II. Specific Embodiments

[0018] A. BT-R₁ Protein

[0019] Prior to the present invention, although members of the BT-toxinfamily of protein were known, no one had identified the receptor that isbound by these toxin proteins. The present invention provides, in part,the amino acid sequences of a BT-toxin receptor that is expressed in themidgut of Maduca sexta.

[0020] In one embodiment, the present invention provides the ability toisolate or produce a previously unknown protein by using knownpurification methods, the cloned nucleic acid molecules herein describedor by synthesizing a protein having the amino acid sequence hereindisclosed.

[0021] As used herein, BT-R₁ refers to a protein that has the amino acidsequence of BT-R₁ provided in FIG. 1, as well as allelic variants of theBT-R₁ sequence, and conservative substitutions mutants of the BT-R₁sequence that have BT-R₁ activity. BT-R₁ is comprised of a singlesubunit, has a molecular weight of 210 kD, and has the amino acidsequence provided in FIG. 1. A prediction of the structure of BT-R₁ isprovided in FIG. 3.

[0022] The BT-R₁ protein of the present invention includes thespecifically identified and characterized variant herein described, aswell as allelic variants, conservative substitution variants andhomologues (FIG. 7) that can be isolated/generated and characterizedwithout undue experimentation following the methods outlined below. Forthe sake of convenience, all BT-R₁ proteins will be collectivelyreferred to as the BT-R₁ proteins, the BT-R₁ proteins of the presentinvention or BT-R₁.

[0023] The term “BT-R₁” includes all naturally occurring allelicvariants of the Manduca sexta BT-R₁ protein provided in FIG. 1. Ingeneral, naturally occurring allelic variants of Manduca sexta BT-R₁will share significant homology, at least 75%, and generally at least90%, to the BT-R₁ amino acid sequence provided in Seq. ID No:2. Allelicvariants, though possessing a slightly different amino acid sequencethan Seq. ID No:2, will be expressed as a transmembrane protein in thedigestive tract of an insect or other organism. Typically, allelicvariants of the BT-R₁ protein will contain conservative amino acidsubstitutions from the BT-R₁ sequence herein described or will contain asubstitution of an amino acid from a corresponding position in a BT-R₁homologue (a BT-R₁ protein isolated from an organism other than Manducasexta).

[0024] One class of BT-R₁ allelic variants will be proteins that share ahigh degree of homology with at least a small region of the amino acidsequence provided in Seq. ID No:_, but may further contain a radicaldeparture from the sequence, such as a nonconservative substitution,truncation, insertion or frame shift. Such alleles are termed mutantalleles of BT-R₁ and represent proteins that typically do not performthe same biological functions as does the BT-R₁ variant of Seq. ID No:2.

[0025] The BT-R₁ proteins of the present invention are preferably inisolated form. As used herein, a protein is said to be isolated whenphysical, mechanical or chemical methods are employed to remove theBT-R₁ protein from cellular constituents that are normally associatedwith the protein. A skilled artisan can readily employ standardpurification methods to obtain an isolated BT-R₁ protein. The nature anddegree of isolation will depend on the intended use.

[0026] The cloning of the BT-R₁ encoding nucleic acid molecule makes itpossible to generate defined fragments of the BT-R₁ proteins of thepresent invention. As discussed below, fragments of BT-R₁ areparticularly useful in: generating domain specific antibodies;identifying agents that bind to toxin binding domain on BT-R₁;identifying toxin-binding structures; identifying cellular factors thatbind to BT-R₁; isolating homologues or other allelic forms of BT-R₁; andstudying the mode of action of BT-toxins.

[0027] Fragments of the BT-R₁ proteins can be generated using standardpeptide synthesis technology and the amino acid sequence of Manducasexta BT-R₁ disclosed herein. Alternatively, as illustrated in Example5, recombinant methods can be used to generate nucleic acid moleculesthat encode a fragment of the BT-R₁ protein. Fragments of the BT-R₁protein subunits that contain particularly interesting structures can beidentified using art-known methods such as by using an immunogenicityplot, Chou-Fasman plot, Garnier-Robson plot, Kyte-Doolittle plot,Eisenberg plot, Karplus-Schultz plot or Jameson-Wolf plot of the BT-R₁protein. Fragments containing such residues are particularly useful ingenerating domain specific anti-BT-R₁ antibodies or in identifyingcellular factors that bind to BT-R₁. One particular fragment that ispreferred for use in identifying insecticidal agents is a solublefragment of BT-R₁ that can bind to a member of the BT family of toxins.In Example 5, a fragment of BT-R₁ that binds to a BT-toxin is disclosed.

[0028] As described below, members of the BT-R₁ family of proteins canbe used for, but are not limited to: 1) a target to identify agents thatbind to BT-R₁, 2) a target or bait to identify and isolate bindingpartners and cellular factors that bind to BT-R₁, 3) an assay target toidentify BT-R₁ and other receptor-mediated activity, and 4) a marker ofcells that express a member of the BT-R₁ family of proteins.

[0029] B. Anti-BT-R₁ Antibodies

[0030] The present invention further provides antibodies that bindBT-R₁. The most preferred antibodies will selectively bind to BT-R₁ andwill not bind (or will only bind weakly) to non-BT-R₁ proteins.Anti-BT-R₁ antibodies that are especially contemplated includemonoclonal and polyclonal antibodies as well as fragments containing theantigen binding domain and/or one or more complement determining regions(CDRs) of these antibodies.

[0031] Antibodies are generally prepared by immunizing a suitablemammalian host using a BT-R₁ protein (synthetic or isolated), orfragment, in isolated or immunoconjugated form (Harlow, Antibodies, ColdSpring Harbor Press, NY (1989)). Regions of the BT-R₁ protein that showimmunogenic structure can readily be identified using art-known methods.Other important regions and domains can readily be identified usingprotein analytical and comparative methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis. Fragments containing these residues areparticularly suited in generating specific classes of anti-BT-R₁antibodies. Particularly useful fragments include, but are not limitedto, the BT-toxin binding domain of BT-R₁ identified in Example 5.

[0032] Methods for preparing a protein for use as an immunogen and forpreparing immunogenic conjugates of a protein with a carrier such asBSA, KLH, or other carrier proteins are well known in the art. In somecircumstances, direct conjugation with reagents such as carbodiimide maybe used; in other instances linking reagents like those supplied byPierce Chemical Co., Rockford, Ill., may be effective.

[0033] Administration of a BT-R₁ immunogen is conducted generally byinjection over a suitable time period in combination with a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

[0034] Although the polyclonal antisera produced in this way may besatisfactory for some applications, for many other applications,monoclonal antibody preparations are preferred. Immortalized cell lineswhich secrete a desired monoclonal antibody may be prepared using thestandard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the BT-R₁ protein orBT-R₁ fragment. When the appropriate immortalized cell culture secretingthe desired antibody is identified, the cells can be cultured either invitro or by production in ascites fluid.

[0035] The desired monoclonal antibodies are then recovered from theculture supernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)₂ fragments is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

[0036] The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the BT-R₁ protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin.

[0037] As described below, anti-BT-R₁ antibodies are useful asmodulators of BT-R₁ activity, are useful in in vitro and in vivoantibody based assays methods for detecting BT-R₁ expression/activity,in generating toxin conjugates, for purifying homologues of Manducasexta BT-R₁ in generating anti-ideotypic antibodies that mimic the BT-R₁protein and in identifying competitive inhibitors of BT-toxin/BT-R₁interactions.

[0038] C. BT-R₁ Encoding Nucleic Acid Molecules

[0039] As described above, the present invention is based, in part, onisolating nucleic acid molecules from Manduca sexta that encode BT-R₁.Accordingly, the present invention further provides nucleic acidmolecules that encode the BT-R₁ protein, as herein defined, preferablyin isolated form. For convenience, all BT-R₁ encoding nucleic acidmolecules will be referred to as BT-R₁ encoding nucleic acid molecules,the BT-R₁ genes, or BT-R₁. The nucleotide sequence of the Manduca sextanucleic acid molecule that encodes one allelic form of BT-R₁ is providedin FIG. 1.

[0040] As used herein, a “nucleic acid molecule” is defined as an RNA orDNA molecule that encodes a peptide as defined above, or iscomplementary to a nucleic acid sequence encoding such peptides.Particularly preferred nucleic acid molecules will have a nucleotidesequence identical to or complementary to the Manduca sexta DNAsequences herein disclosed. Specifically contemplated are genomic DNA,cDNAs, synthetically prepared DNAs, and antisense molecules, as well asnucleic acids based on an alternative backbone or including alternativebases, whether derived from natural sources or synthesized. A skilledartisan can readily obtain these classes of nucleic acid molecules usingthe herein described BT-R₁ sequences. However, such nucleic acidmolecules, are defined further as being novel and unobvious over anyprior art nucleic acid molecules encoding non-BT-R₁ proteins. Forexample, the BT-R₁ sequences of the present invention specificallyexcludes previously identified nucleic acid molecules that share onlypartial homology to BT-R₁. Such excluded sequences include identifiedmembers of the cadhedrin family of proteins.

[0041] As used herein, a nucleic acid molecule is said to be “isolated”when the nucleic acid molecule is substantially separated fromcontaminant nucleic acid molecules that encode polypeptides other thanBT-R₁. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain qn isolated BT-R₁ encoding nucleic acid molecule.

[0042] The present invention further provides fragments of the BT-R₁encoding nucleic acid molecules of the present invention. As usedherein, a fragment of a BT-R₁ encoding nucleic acid molecule refers to asmall portion of the entire BT-R₁ sequence. The size of the fragmentwill be determined by its intended use. For example, if the fragment ischosen so as to encode the toxin binding domain of BT-R₁ identified inExample 5, then the fragment will need to be large enough to encode thetoxin binding domain of the BT-R₁ protein. If the fragment is to be usedas a nucleic acid probe or PCR primer, then the fragment length ischosen so as to obtain a relatively small number of false positivesduring probing/priming. Fragments of the Manduca sexta BT-R₁ gene thatare particularly useful as selective hybridization probes or PCR primerscan be readily identified from the entire BT-R₁ sequence using art-knownmethods.

[0043] Another class of fragments of BT-R₁ encoding nucleic acidmolecules are the expression control sequence found upstream anddownstream from the BT-R₁ encoding region found in genomic clones of theBT-R₁ gene. Specifically, tissue and developmental specific expressioncontrol elements can be identified as being 5′ to the BT-R₁ encodingregion found in genomic clones of the BT-R₁ gene. Such expressioncontrol sequence are useful in generating expression vectors forexpressing genes in the digestive tract of a transgenic organism. Asdescribed in more detail below, a skilled artisan can readily use theBT-R₁ cDNA sequence herein described to isolate and identify genomicBT-R₁ sequences and the expression control elements found in the BT-R₁gene.

[0044] Fragments of the BT-R₁ encoding nucleic acid molecules of thepresent invention (i.e., synthetic oligonucleotides) that are used asprobes or specific primers for the polymerase chain reaction (PCR), orto synthesize gene sequences encoding BT-R₁ proteins, can easily besynthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci, et al., J Am Chem Soc (1981) 103:3185-3191, orusing automated synthesis methods. In addition, larger DNA segments canreadily be prepared by well known methods, such as synthesis of a groupof oligonucleotides that define various modular segments of the BT-R₁gene, followed by ligation of oligonucleotides to build the completemodified BT-R₁ gene.

[0045] The BT-R₁ encoding nucleic acid molecules of the presentinvention may further be modified so as to contain a detectable labelfor diagnostic and probe purposes. As described above, such probes canbe used to identify nucleic acid molecules encoding other allelicvariants or homologues of the BT-R₁ proteins and as described below,such probes can be used to identify the presence of a BT-R₁ protein as ameans for identifying cells that express a BT-R₁ protein. A variety ofsuch labels are known in the art and can readily be employed with theBT-R₁ encoding molecules herein described. Suitable labels include, butare not limited to, biotin, radiolabeled nucleotides, biotin, and thelike. A skilled artisan can employ any of the art-known labels to obtaina labeled BT-R₁ encoding nucleic acid molecule.

[0046] D. Isolation of Other BT-R₁ Encoding Nucleic Acid Molecules

[0047] The identification of the BT-R₁ protein from Manduca sexta andthe corresponding encoding nucleic acid molecules, has made possible theidentification of and isolation of: 1) BT-R₁ proteins from organismsother than Manduca sexta, hereinafter referred to collectively as BT-R₁homologues, 2) other allelic and mutant forms of the Manduca sexta BT-R₁protein (described above), and 3) the corresponding genomic DNA thatcontains the BT-R₁ gene. The most preferred source of BT-R₁ homologuesare insects, the most preferred being members of the Lepidopteran,Coleopteran and Dipteran orders of insects. Evidence of the existence ofBT-R₁ homologues is provided in FIG. 7.

[0048] Essentially, a skilled artisan can readily use the amino acidsequence of the Manduca sexta BT-R₁ protein to generate antibody probesto screen expression libraries prepared from cells and organisms.Typically, polyclonal antiserum from mammals such as rabbits immunizedwith the purified protein (as described above) or monoclonal antibodiescan be used to probe an expression library, prepared from a targetorganism, to obtain the appropriate coding sequence for a BT-R₁homologue. The cloned cDNA sequence can be expressed as a fusionprotein, expressed directly using its own control sequences, orexpressed by constructing an expression cassette using control sequencesappropriate to the particular host used for expression of the enzyme.

[0049] Alternatively, a portion of the BT-R₁ encoding sequence hereindescribed can be synthesized and used as a probe to retrieve DNAencoding a member of the BT-R₁ family of proteins from organisms otherthan Manduca sexta, allelic variants of the Manduca sexta BT-R₁ proteinherein described, and genomic sequence containing the BT-R₁ gene.Oligomers containing approximately 18-20 nucleotides (encoding about a6-7 amino acid stretch) are prepared and used to screen genomic DNA orcDNA libraries to obtain hybridization under stringent conditions orconditions of sufficient stringency to eliminate an undue level of falsepositives.

[0050] Additionally, pairs of oligonucleotide primers can be preparedfor use in a polymerase chain reaction (PCR) to selectivelyamplify/clone a BT-R₁-encoding nucleic acid molecule, or fragmentthereof. A PCR denature/anneal/extend cycle for using such PCR primersis well known in the art and can readily be adapted for use in isolatingother BT-R₁ encoding nucleic acid molecules. Regions of the Manducasexta BT-R₁ gene that are particularly well suited for use as a probe oras primers can be readily identified by one skilled in the art.

[0051] Non-Manduca sexta homologues of BT-R₁, naturally occurringallelic variants of the Manduca sexta BT-R₁ gene and genomic BT-R₁sequences will share a high degree of homology to the Manduca sextaBT-R₁ sequence herein described. In general, such nucleic acid moleculeswill hybridize to the Manduca sexta BT-R₁ sequence under highstringency. Such sequences will typically contain at least 70% homology,preferably at least 80%, most preferably at least 90% homology to theManduca sexta BT-R₁ sequence of Seq. ID No:1.

[0052] In general, nucleic acid molecules that encode homologues of theManduca sexta BT-R₁ protein will hybridize to the Manduca sexta BT-R₁sequence under stringent conditions. “Stringent conditions” are thosethat (1) employ low ionic strength and high temperature for washing, forexample, 0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50° C., or (2)employ during hybridization a denaturing agent such as formamide, forexample, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM NaCl, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5×SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1%SDS. A skilled artisan can readily determine and vary the stringencyconditions appropriately to obtain a clear and detectable hybridizationsignal.

[0053] The presence of similar receptors in noninsect organisms as wellas other insects besides those harboring BT-R₁ is supported by thesequence similarity of the BT-R₁ protein to that of the various membersof the cadherin superfamily of proteins, which are membraneglycoproteins believed to mediate calcium-dependent cell aggregation andsorting. See, for example, Takeichi, M. Science (1991) 251:1451; andTakeichi, M. N Rev Biochem (1990) 59:237.

[0054] Included in this superfamily are desmoglien, desmocollins, theDrosophila fat tumor suppressor, Manduca sexta intestinal peptidetransport protein and T-cadherin. All of these proteins share commonextracellular motifs although their cytoplasmic domains differ. Goodwin,L. et al. Biochem Biophys Res Commun (1990) 173:1224; Holton, J. L. etal. J Cell Sci (1990) 97:239; Bestal, D. J. J Cell Biol (1992) 119:451;Mahoney, P. A. et al. Cell (1991) 853; Dantzig, A. H. et al. Science(1994) 264:430; and Sano, K. et al. EMBO J (1993) 12:2249. Inclusion ofBT-R₁ in the cadherin superfamily is further supported by the reportthat EDTA decreases the binding of CryIAb toxin of BT to the 210 kDreceptor of M. sexta (Martinez-Ramirez, A. C. et al. Biochm Biophys ResCommun (1994) 201:782).

[0055] It is noted below that the amino acid sequence of BT-R₁ revealsthat a calciumbinding motif is present. This is consistent with thepossibility that cells having receptors to bind toxin may themselvessurvive although they render the tissues in which they are includedpermeable to solutes and thus effect disintegration of the tissue. Sucha mechanism is proposed for the death of insects that ingest the toxinvia the epithelial cells in their midgut by Knowles, B. H. et al.Biochim Biophys Acta (1987) 924:509. Such a mechanism is also supportedin part by the results set forth in Example 4 hereinbelow which indicatethat the effect of the toxin on embryonic 293 cells modified to expressthe receptor at their surface is reversible.

[0056] E. rDNA Molecules Containing a BT-R₁ Encoding Nucleic AcidMolecule

[0057] The present invention further provides recombinant DNA molecules(rDNAs) that contain a BT-R₁ encoding sequences as herein described, ora fragment thereof, such as a soluble fragment of BT-R₁ that containsthe BT-toxin binding site. As used herein, a rDNA molecule is a DNAmolecule that has been subjected to molecular manipulation in vitro.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning (1989). In the preferredrDNA molecules of the present invention, a BT-R₁ encoding DNA sequencethat encodes a BT-R₁ protein or a fragment of BT-R₁, is operably linkedto one or more expression control sequences and/or vector sequences.

[0058] The choice of vector and/or expression control sequences to whichthe BT-R₁ encoding sequence is operably linked depends directly, as iswell known in the art, on the functional properties desired, e.g.,protein expression, and the host cell to be transformed. A vectorcontemplated by the present invention is at least capable of directingthe replication or insertion into the host chromosome, and preferablyalso expression, of the BT-R₁ encoding sequence included in the rDNAmolecule.

[0059] Expression control elements that are used for regulating theexpression of an operably linked protein encoding sequence are known inthe art and include, but are not limited to, inducible promoters,constitutive promoters, secretion signals, enhancers, transcriptionterminators and other regulatory elements. Preferably, an induciblepromoter that is readily controlled, such as being responsive to anutrient in the host cell's medium, is used. Further, for solublefragments, it may be desirable to use secretion signals to direct thesecretion of the BT-R₁ protein, or fragment, out of the cell.

[0060] In one embodiment, the vector containing a BT-R₁ encoding nucleicacid molecule will include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule intrachromosomally in a prokaryotic hostcell, such as a bacterial host cell, transformed therewith. Suchreplicons are well known in the art. In addition, vectors that include aprokaryotic replicon may also include a gene whose expression confers adetectable marker such as a drug resistance. Typical bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline.

[0061] Vectors that include a prokaryotic replicon can further include aprokaryotic or viral promoter capable of directing the expression(transcription and translation) of the BT-R₁ encoding sequence in abacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Typical of such vector plasmids are pUC8,pUC9, pBR322 and pBR329 available from Biorad Laboratories (Richmond,Calif.), pPL and pKK223 available from Pharmacia, Piscataway, N.J.

[0062] Expression vectors compatible with eukaryotic cells, preferablythose compatible with vertebrate cells, can also be used to variant rDNAmolecules that contain a BT-R₁ encoding sequence. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC,#31255), the vector pCDM8 described herein, and the like eukaryoticexpression vectors.

[0063] Eukaryotic cell expression vectors used to construct the rDNAmolecules of the present invention may further include a selectablemarker that is effective in an eukaryotic cell, preferably a drugresistance selection marker. A preferred drug resistance marker is thegene whose expression results in neomycin resistance, i.e., the neomycinphosphotransferase (neo) gene. Southern et al., J Mol Anal Genet (1982)1:327-341. Alternatively, the selectable marker can be present on aseparate plasmid, and the two vectors are introduced by cotransfectionof the host cell, and selected by culturing in the presence of theappropriate drug for the selectable marker.

[0064] F. Host Cells Containing an Exogenously Supplied BT-R₁ EncodingNucleic Acid Molecule

[0065] The present invention further provides host cells transformedwith a nucleic acid molecule that encodes a BT-R₁ protein of the presentinvention, either the entire BT-R₁ protein or a fragment thereof. Thehost cell can be either prokaryotic or eukaryotic. Eukaryotic cellsuseful for expression of a BT-R₁ protein are not limited, so long as thecell line is compatible with cell culture methods and compatible withthe propagation of the expression vector and expression of a BT-R₁ gene.Preferred eukaryotic host cells include, but are not limited to, yeast,insect and mammalian cells, the most preferred being cells that do notnatiurally express a BT-R₁ protein

[0066] Any prokaryotic host can be used to express a BT-R₁-encoding rDNAmolecule. The preferred prokaryotic host is E. coli.

[0067] Transformation of appropriate cell hosts with an rDNA molecule ofthe present invention is accomplished by well known methods thattypically depend on the type of vector used and host system employed.With regard to transformation of prokaryotic host cells, electroporationand salt treatment methods are typically employed, see, for example,Cohen et al., Proc Acad Sci USA (1972) 69:2110; and Maniatis et al.,Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). With regard to transformation ofvertebrate cells with vectors containing rDNAs, electroporation,cationic lipid or salt treatment methods are typically employed, see,for example, Graham et al., Virol (1973) 52:456; Wigler et al., ProcNatl Acad Sci USA (1979) 76:1373-76.

[0068] Successfully transformed cells, i.e., cells that contain an rDNAmolecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anrDNA of the present invention can be cloned to produce single colonies.Cells from those colonies can be harvested, lysed and their DNA contentexamined for the presence of the rDNA using a method such as thatdescribed by Southern, J Mol Biol (1975) 98:503, or Berent et al.,Biotech (1985) 3:208 or the proteins produced from the cell assayed viaan immunological method.

[0069] G. Production of a BT-R₁ Protein Using an rDNA Molecule

[0070] The present invention further provides methods for producing aBT-R₁ protein that uses one of the BT-R₁ encoding nucleic acid moleculesherein described. In general terms, the production of a recombinantBT-R₁ protein typically involves the following steps.

[0071] First, a nucleic acid molecule is obtained that encodes a BT-R₁protein or a fragment thereof, such as the nucleic acid moleculedepicted in FIG. 1. The BT-R₁ encoding nucleic acid molecule is thenpreferably placed in an operable linkage with suitable controlsequences, as described above, to generate an expression unit containingthe BT-R₁ encoding sequence. The expression unit is used to transform asuitable host and the transformed host is cultured under conditions thatallow the production of the BT-R₁ protein. Optionally the BT-R₁ proteinis isolated from the medium or from the cells; recovery and purificationof the protein may not be necessary in some instances where someimpurities may be tolerated.

[0072] Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in an appropriate host. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using an appropriate combination of replicons and controlsequences. The control sequences, expression vectors, and transformationmethods are dependent on the type of host cell used to express the geneand were discussed in detail earlier. Suitable restriction sites can, ifnot normally available, be added to the ends of the coding sequence soas to provide an excisable gene to insert into these vectors. A skilledartisan can readily adapt any host/expression system known in the artfor use with BT-R₁ encoding sequences to produce a BT-R₁ protein.

[0073] H. Identification of Agents and Cellular Constituents that Bindto a BT-R₁ Protein

[0074] Another embodiment of the present invention provides methods foridentifying agents and cellular constituents that bind to BT-R₁.Specifically, agents and cellular constituents that bind to BT-R₁ can beidentified by: 1) the ability of the agent/constituent to bind to BT-R₁,2) the ability to block BT-toxin binding to BT-R₁, and/or 3) the abilityto kill BT-R₁ expressing cells. Activity assays for BT-R₁ activity andbinding and competitive assays using a BT-R₁ protein are suitable foruse in high through-put screening methods, particularly using a solublefragment of BT-R₁ that contains the BT-toxin binding domain. such asthat disclosed in Example 5.

[0075] In detail, in one embodiment, BT-R₁ is mixed with an agent orcellular extract. After mixing under conditions that allow associationof BT-R₁ with the agent or component of the extract, the mixture isanalyzed to determine if the agent/component bound to the BT-R₁. Bindingagents/components are identified as being able to bind to BT-R₁.Alternatively or consecutively, BT-R₁ activity can be directly assessedas a means for identifying agonists and antagonists of BT-R₁ activity.

[0076] Alternatively, targets that are bound by a BT-R₁ protein can beidentified using a yeast two-hybrid system or using a binding-captureassay. In the yeast two hybrid system, an expression unit encoding afusion protein made up of one subunit of a two subunit transcriptionfactor and the BT-R₁ protein is introduced and expressed in a yeastcell. The cell is further modified to contain 1) an expression unitencoding a detectable marker whose expression requires the two subunittranscription factor for expression and 2) an expression unit thatencodes a fusion protein made up of the second subunit of thetranscription factor and a cloned segment of DNA. If the cloned segmentof DNA encodes a protein that binds to the BT-R₁ protein, the expressionresults in the interaction of the BT-R₁ and the encoded protein. Thisbrings the two subunits of the transcription factor into bindingproximity, allowing reconstitution of the transcription factor. Thisresults in the expression of the detectable marker. The yeast two hybridsystem is particularly useful in screening a library of cDNA encodingsegments for cellular binding partners of BT-R₁.

[0077] The BT-R₁ protein used in the above assays can be: an isolatedand fully characterized protein, a fragment of a BT-R₁ protein (such asa soluble fragment containing the BT-toxin binding site), a cell thathas been altered to express a BT-R₁ protein/fragment or a fraction of acell that has been altered to express a BT-R₁ protein/fragment. Further,the BT-R₁ protein can be the entire BT-R₁ protein or a defined fragmentof the BT-R₁ protein. It will be apparent to one of ordinary skill inthe art that so long as the BT-R₁ protein or fragment can be assayed foragent binding, e.g., by a shift in molecular weight or activity, thepresent assay can be used.

[0078] The method used to identify whether an agent/cellular componentbinds to a BT-R₁ protein will be based primarily on the nature of theBT-R₁ protein used. For example, a gel retardation assay can be used todetermine whether an agent binds to BT-R₁ or a fragment thereof.Alternatively, immunodetection and biochip technologies can be adoptedfor use with the BT-R₁ protein. A skilled artisan can readily employnumerous art-known techniques for determining whether a particular agentbinds to a BT-R₁ protein.

[0079] Agents and cellular components can be further, or alternatively,tested for the ability to block the binding of a BT-toxin to a BT-R₁protein/fragment. Alternatively, antibodies to the BT-toxin binding siteor other agents that bind to the BT-toxin binding site on the BT-R₁protein can be used in place of the BT-toxin.

[0080] Agents and cellular components can be further tested for theability to modulate the activity of a BT-R₁ protein using a cell-freeassay system or a cellular assay system. As the activities of the BT-R₁protein become more defined, functional assays based on the identifiedactivity can be employed.

[0081] As used herein, an agent is said to antagonize BT-R₁ activitywhen the agent reduces BT-R₁ activity. The preferred antagonist willselectively antagonize BT-R₁, not affecting any other cellular proteins.Further, the preferred antagonist will reduce BT-R₁ activity by morethan 50%, more preferably by more than 90%, most preferably eliminatingall BT-R₁ activity.

[0082] As used herein, an agent is said to agonize BT-R₁ activity whenthe agent increases BT-R₁ activity. The preferred agonist willselectively agonize BT-R₁, not affecting any other cellular proteins.Further, the preferred antagonist will increase BT-R₁ activity by morethan 50%, more preferably by more than 90%, most preferably more thandoubling BT-R₁ activity.

[0083] Agents that are assayed in the above method can be randomlyselected or rationally selected or designed. As used herein, an agent issaid to be randomly selected when the agent is chosen randomly withoutconsidering the specific sequences of the BT-R₁ protein or BT-toxin. Anexample of randomly selected agents is the use of a chemical library ora peptide combinatorial library, or a growth broth of an organism orplant extract.

[0084] As used herein, an agent is said to be rationally selected ordesigned when the agent is chosen on a nonrandom basis that takes intoaccount the sequence of the target site and/or its conformation inconnection with the agent's action. Agents can be rationally selected orrationally designed by utilizing the peptide sequences that make up theBT-R₁ protein and BT-toxin. For example, a rationally selected peptideagent can be a peptide whose amino acid sequence is identical to afragment of a BT-R₁ protein or BT-toxin.

[0085] The agents tested in the methods of the present invention can be,as examples, peptides, small molecules, and vitamin derivatives, as wellas carbohydrates. A skilled artisan can readily recognize that there isno limit as to the structural nature of the agents used in the presentscreening method. One class of agents of the present invention arepeptide agents whose amino acid sequences are chosen based on the aminoacid sequence of the BT-R₁ protein or BT-toxin. Small peptide agent, canserve as competitive inhibitors of BT-R₁ protein activity.

[0086] Peptide agents can be prepared using standard solid phase (orsolution phase) peptide synthesis methods, as is known in the art. Inaddition, the DNA encoding these peptides may be synthesized usingcommercially available oligonucleotide synthesis instrumentation andproduced recombinantly using standard recombinant production systems.The production using solid phase peptide synthesis is necessitated ifnon-gene-encoded amino acids are to be included.

[0087] Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of the BT-R₁ protein. Asdescribed above, antibodies are obtained by immunization of suitablemammalian subjects with peptides, containing as antigenic regions, thoseportions of the BT-R₁ protein intended to be targeted by the antibodies.Critical regions particularly include the BT-toxin binding domainidentified in Example 5. Such agents can be used in competitive bindingstudies to identify second generation BT-R₁ binding agents.

[0088] The cellular extracts tested in the methods of the presentinvention can be, as examples, aqueous extracts of cells or tissues,organic extracts of cells or tissues or partially purified cellularfractions. A skilled artisan can readily recognize that there is nolimit as to the source of the cellular extract used in the screeningmethod of the present invention. The preferred source for isolatingcellular binding partners of BT-R₁ are cells that express BT-R₁ or cellsthat are in close proximity to BT-R₁ expressing cells.

[0089] An outline of one screening method is as follows. Cells aremodified by transfection, retroviral infection, electroporation or otherknown means, to express a BT-R₁ protein and then cultured underconditions wherein the receptor protein is produced and displayed. Ifdesired, the cells are then recovered from the culture for use in theassay, or the culture itself can be used per se.

[0090] In the assays, the modified cells are contacted with thecandidate toxin and the effect on metabolism or morphology is noted inthe presence and absence of the candidate. The effect may becytotoxic—i.e., the cells may themselves exhibit one of the indices ofcell death, such as reduced thymidine uptake, slower increase in opticaldensity of the culture, reduced exclusion of vital dyes (e.g., trypanblue), increased release of viability markers such as chromium andrubidium, and the like. The differential response between thetoxin-treated cells and the cells absent the toxin is then noted. Thestrength of the toxin can be assessed by noting the strength of theresponse.

[0091] These assays may be conducted directly as described above orcompetitively with known toxins. For example, one approach might be tomeasure the diminution in binding of labeled BT cry toxin in thepresence and absence of the toxin candidate.

[0092] In addition to simply screening candidates, the screen can beused to devise improved forms of toxins which are more specific or lessspecific to particular classes of insects as desired. The ability todetermine binding affinity (K_(a) and K_(d)), dissociation andassociation rates, and cytotoxic effects of a candidate allows quick,accurate and reproducible screening techniques for a large number oftoxins and other ligands under identical conditions which was notpossible heretofore. Such information will facilitate the selection ofthe most effective toxins and ligands for any given receptor obtainedfrom any desired host cell.

[0093] Competition assays may also employ antibodies that arespecifically immunoreactive with the receptor. Such antibodies can beprepared in the conventional manner by administering the purifiedreceptor to a vertebrate animal, monitoring antibody titers andrecovering the antisera or the antibody-producing cells forimmortalization, to obtain immortalized cells capable of secretingantibodies of the appropriate specificity. Techniques for obtainingimmortalized B cells and for screening them for secretion of the desiredantibody are now conventional in the art. The resulting monoclonalantibodies may be more effective than the polyclonal antisera ascompetition reagents; furthermore, the availability of the immortalizedcell line secreting the desired antibody assures uniformity ofproduction of the same reagent over time. The information and thestructural characteristics of toxins and ligands tested will permit arational approach to designing more efficient toxins and ligands.Additionally, such assays will lead to a better understanding of thefunction and the structure/function relationship of both toxin/ligandand BT-R₁ analogs. In turn, this will allow the development of highlyeffective toxins/ligands. Ligands include natural and modified toxins,antibodies (anti-receptor and antiidiotypic antibodies which mimic aportion of a toxin that binds to a receptor, and whatever smallmolecules bind the receptors.

[0094] I. Uses of Agents that Bind to a BT-R₁ Protein

[0095] As provided in the Background section, BT-R₁ is the target forthe insecticidal activity of BT-toxins. Agents that bind a BT-R₁ proteincan be used: 1) to kill BT-R₁ expressing cells, 2) to identify agentsthat block the interaction of a BT-toxin with BT-R₁ and 3) in methodsfor identifying cells that express BT-R₁.

[0096] The methods employed in using the BT-R₁ binding agents will bebased primarily on the nature of the BT-R₁ binding agent and itsintended use. For example, a BT-R₁ binding agent can be used to: delivera conjugated toxin to a BT-R₁ expressing cell; modulate BT-R₁ activity;directly kill BT-R₁ expressing cells; or screen for and identifycompetitive binding agents. An agent that inhibits the activity of BT-R₁can be used to directly inhibit the growth of BT-R₁ expressing cells.Further, identified cellular factors that bind to BT-R₁ can, themselves,be used in binding/competitive assays to identify agonist andantagonists of BT-R₁.

[0097] J. Methods for Identifying the Presence of a BT-R₁ Protein orGene

[0098] The present invention further provides methods for identifyingcells, tissues or organisms expressing a BT-R₁ protein or a BT-R₁ gene.Such methods can be used to diagnose the presence of cells or anorganism that expresses a BT-R₁ protein in vivo or in vitro. The methodsof the present invention are particularly useful in the determining thepresence of cells that are a target for BT-toxin activity or foridentifying susceptibility of an organism to a BT-toxin or BT-toxin-likeagent. Specifically, the presence of a BT-R₁ protein can be identifiedby determining whether a BT-R₁ protein, or nucleic acid encoding a BT-R₁protein, is expressed in a cell, tissue or organism.

[0099] A variety of immunological and molecular genetic techniques canbe used to determine if a BT-R₁ protein is expressed/produced in aparticular cell or sample. In general, an extract containing nucleicacid molecules or an extract containing proteins is prepared. Theextract is then assayed to determine whether a BT-R₁ protein, or a BT-R₁encoding nucleic acid molecule, is produced in the cell.

[0100] For example, to perform a diagnostic test based on nucleic acidmolecules, a suitable nucleic acid sample is obtained and prepared usingconventional techniques. DNA can be prepared, for example, simply byboiling a sample in SDS. The extracted nucleic acid can then besubjected to amplification, for example by using the polymerase chainreaction (PCR) according to standard procedures, such as a RT-PCRmethod, to selectively amplify a BT-R₁ encoding nucleic acid molecule orfragment thereof. The size or presence of a specific amplified fragment(typically following restriction endonuclease digestion) is thendetermined using gel electrophoresis or the nucleotide sequence of thefragment is determined (for example, see Weber and May Am J Hum Genet(1989) 44:388-339; Davies, J. et al. Nature (1994) 371:130-136)). Theresulting size of the fragment or sequence is then compared to the knownBT-R₁ proteins encoding sequences, for example via hybridization probe.Using this method, the presence of a BT-R₁ protein can be identified.

[0101] To perform a diagnostic test based on proteins, a suitableprotein sample is obtained and prepared using conventional techniques.Protein samples can be prepared, for example, simply by mixing a samplewith SDS followed by salt precipitation of a protein fraction. Theextracted protein can then be analyzed to determine the presence of aBT-R₁ protein using known methods. For example, the presence of specificsized or charged variants of a protein can be identified using mobilityin an electric filed. Alternatively, antibodies can be used fordetection purposes. A skilled artisan can readily adapt known proteinanalytical methods to determine if a sample contains a BT-R₁ protein.

[0102] Alternatively, BT-R₁ protein or gene expression can also be usedin methods to identify agents that decrease the level of expression of aBT-R₁ gene. For example, cells or tissues expressing a BT-R₁ protein canbe contacted with a test agent to determine the effects of the agent onBT-R₁ protein/gene expression. Agents that activate BT-R₁ protein/geneexpression can be used as an agonist of BT-R₁ activity whereas agentsthat decrease BT-R₁ protein/gene expression can be used as an antagonistof BT-R₁ activity.

[0103] K. Methods to Sensitize Cells

[0104] The present invention further provides methods of sensitizingcells such that they become susceptible to killing with a BT-toxin, or aBT-toxin analog. Specifically, host cells transformed to express BT-R₁receptor, or a homolog of the BT-R₁ receptor, become sensitive to themode of action of BT-toxins. The binding of a BT-toxin to a BT-R₁receptor expressed on the surface of the transformed cells results ininduction of a cellular death and apoptosis of the cell expressing theBT-R₁ receptor. Accordingly, the BT-R₁ receptor is an appropriatecandidate for use in transforming cells in which it is desirable toinduce cell death.

[0105] There are numerous situations in which it is desirable tointroduce the selected gene into a selected population of cells, thusbringing about cell death. One such example is in the therapeutictreatment of cancer cells. In using specifically targeted vectors fordelivery of BT-R₁-encoding DNA molecules into a tumor cell, tumor cellswithin a patient can be engineered to express a BT-R₁ protein. Suchcells then become susceptible to death upon treatment with a BT-toxin.Since BT-toxin is not normally toxic to mammalian cells, this method isparticularly applicable to inducing cell death in particular cells in amammalian host. Other situations where it may be desirable to stimulatecell death in particular cells or cell lines are in the treatment ofautoimmune disorders and in the treatment of cells harboring pathogens,such as malaria or HIV agents.

[0106] The choice of the actual steps employed to introduce aBT-R₁-encoding DNA molecule into a cell to render the cells susceptibleto treatment with BT-toxin is based primarily on the cell type that isto be altered, the conditions under which the cell type will be altered,and the overall use envisioned. A skilled artisan can readily adaptart-known methods for use with the BT-R₁-encoding DNA molecule of thepresent invention.

[0107] L. Animal Models and Gene Therapy

[0108] The BT-R₁ gene and the BT-R₁ protein can also serve as a targetfor generating transgenic organisms in which the pattern of BT-R₁expression has been altered. For example, in one application, BT-R₁deficient insects or insect cells can be generated using standardknock-out procedures to inactivate a BT-R₁ gene, or, if such animals arenonviable, inducible BT-R₁ antisense molecules can be used to regulateBT-R₁ activity/expression. Alternatively, cells or an organism can bealtered so as to contain a Manduca sexta BT-R₁ encoding nucleic acidmolecule or an antisense-BT-R₁ expression unit that directs theexpression of a BT-R₁ protein or an antisense molecule in a tissuespecific fashion. In such uses, an organism or cells, for exampleinsects or insect cells, is generated in which the expression of a BT-R₁gene is altered by inactivation or activation and/or replaced by aManduca sexta BT-R₁ gene. This can be accomplished using a variety ofart-known procedures such as targeted recombination. Once generated, theBT-R₁ expression altered cells or organisms can be used to 1) identifybiological and pathological processes mediated by the BT-R₁ protein, 2)identify proteins and other genes that interact with the BT-R₁ protein,3) identify agents that can be exogenously supplied to overcome a BT-R₁protein deficiency and 4) serve as an appropriate screen for identifyingmutations within the BT-R₁ gene that increases or decreases activity.

[0109] For example, it is possible to generate transgenic insects, suchas members of the dipteran order, expressing the Manduca sexta minigeneencoding BT-R₁ in a tissue specific-fashion and test the effect ofover-expression of the protein in tissues and cells that normally do notcontain the BT-R₁ protein.

[0110] M. Use of Expression Control Elements of the BT-R₁ Gene

[0111] The present invention further provides the expression controlsequences found 5′ of the of the newly identified BT-R₁ gene in a formthat can be used in generating expression vectors. Specifically, theBT-R₁ expression control elements, such as the BT-R₁ promoter, that canreadily be identified as being 5′ from the ATG start codon in the BT-R₁gene, can be used to direct the expression of an operably linked proteinencoding DNA sequence. Since BT-R₁ expression is mostly tissue-specific,the expression control elements are particularly useful in directing theexpression of an introduced transgene in a tissue specific fashion. Askilled artisan can readily use the BT-R₁ gene promoter and otherregulatory elements to generate expression vectors using methods knownin the art.

[0112] Without further description, it is believed that one of ordinaryskill in the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure.

EXAMPLE 1 Purification and Sequence Determination of BT-R₁ Protein

[0113] Midguts of M. sexta were extracted and the BT-R₁ protein purifiedaccording to the method of Vadlamudi, R. K. et al. J Biol Chem (1993)268:1233, referenced above and incorporated herein by reference. Theelectroeluted band was confirmed to contain BT-R₁ protein by binding to¹²⁵I-cryIAb toxin. In gel electrophoresis, the protein bound to toxinhad an apparent weight of approximately 210 kD under reducing andnonreducing conditions.

[0114] The purified electroeluted BT-R₁ was subjected to cyanogenbromide digestion and the cyanogen bromide fragments separated on a 17%high-resolution tricine SDS-polyacrylamide gel as described by Schagger,H. et al. Anal Biochem (1987) 166:368. The separated fragments weretransferred to Problott membranes (Applied Biosystems) and five bandswere extracted and subjected to microsequencing using standardinstrumentation. The amino acid sequences obtained were:

[0115] 1.(Met)-Leu-Asp-Tyr-Glu-Val-Pro-Glu-Phe-Gln-Ser-Ile-Thr-Ile-Arg-Val-Val-Ala-Thr-Asp-Asn-Asn-Asp-Thr-Arg-His-Val-Gly-Val-Ala;

[0116] 2.(Met)-X-Glu-Thr-Tyr-Glu-Leu-Ile-Ile-His-Pro-Phe-Asn-Tyr-Tyr-Ala;

[0117] 3. (Met)-X-X-X-His-Gln-Leu-Pro-Leu-Ala-Gln-Asp-Ile-Lys-Asn-His;

[0118] 4. (Met)-Phe/Pro-Asn/Ile-Val-Arg/Tyr-Val-Asp-Ile/Gly;

[0119] 5. (Met)-Asn-Phe-Phe/His-Ser-Val-Asn-Arg/Asp-Glu.

EXAMPLE 2 Recovery of cDNA

[0120] An M. sexta cDNA library was constructed from midgut tissue inλgt10 using the Superscript Choice System according to themanufacturer's instructions (Life Technologies, Inc.). Degenerateoligonucleotide probes were constructed based on the peptide sequencesdetermined in Example 1 using the methods and approach described inZhang, S. et al. Gene (1991) 105:61. Synthetic oligonucleotidescorresponding to peptides 1-3 of Example 1 were labeled with α³²P usingpolynucleotide kinase and used as probes as described in the standardcloning manual of Maniatis, T. et al. Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2nd ed.1989). A clone hybridizing to all three probes identified from 40positive clones as, hybridizing to all three of the probes wasplaque-purified from a screen of 4×10⁵ recombinants and subcloned intopBluescript (Stratagene). It contained an insert of 5571 bp.

[0121] Double-stranded cDNA in pBluescript was sequenced in bothdirections by the dideoxy termination method with Sequanase (USB)according to the manufacturer's instructions. The sequencing showed anopen reading frame of 4584 base pairs or 1528 amino acids along with apolyadenylation signal at position 5561. The sequence obtained and thededuced amino acid sequence is shown in FIG. 1.

[0122] Thus, the deduced protein has a molecular mass of 172 kD and a pIof approximately 4.5. The amino acid sequences of the cyanogen bromidefragments of native receptor match perfectly within the deduced aminoacid sequence. The open reading frame begins with an ATG that is flankedby the consensus translation initiation sequence GAGATGG for eucaryoticmRNAs as described by Kozak, M. Nucleic Acids Res (1987) 15:8125.

[0123] As shown in FIG. 1, the deduced amino acid sequence includes aputative signal, shown underlined, preceding the mature N-terminusAsn-Glu-Arg-etc. Eleven repeats (cad1-cad11) are shown in theextracellular region upstream of the membrane domain, shown with theheavy underline, at positions 1406-1427. The end of the 11th repeat isshown with an arrowhead. The positions of the five CNBR fragments arealso shown under the complete sequence.

[0124]FIG. 2 compares the BT-R₁ sequence obtained herein with othermembers of the cadherin family. Like known cadherins, the externaldomain of BT-R₁ is highly repetitive and contains 11 repeats(cad1-cad11; see FIG. 2A). The other cadherins compared in FIG. 2B aremouse P cadherin (mP EC1); Drosophila fat EC18 (fat EC18) andprotocadherin (PC42 EC2), and Manduca sexta intestinal transporter(HPT-1-EC-1). The eleven repeats of the cadherin motif in BT-R₁(cad1-cad11) are individually aligned with a single motif sequence fromeach of the other members of the cadherin family.

[0125] Conserved residues are boxed The greatest similarity of BT-R₁ tothe cadherins is with the extracellular repeats of the cadherin motif ofmouse P-cadherin, Drosophila fat tumor suppressor and theprotocadherins, although homologies are not high (20-40 homology and30-60 percent similarity). The conserved repeats of BT-R₁ includedAXDXD, DXE, DXNDXXP, one glutamic acid residue and two glycine residues(FIG. 2B). Motifs A/VXDXD, DXNDN are the consensus sequences for calciumbinding and two such regions are present in a typical cadherin repeat.In all repeats of BT-R₁, the sequence DXNDN is preceded by 8 to 14hydrophobic amino acids. Similar hydrophobic sequences also have beenobserved in the cadherins. The length of the hydrophobic stretchessuggests that these areas are not transmembrane regions buy that therepresent J-sheet structures commonly present in cadherin-like repeats.BT-R₁ contains a putative cytoplasmic domain of 101 amino acids, smallerthan vertebrate cadherin cytoplasmic domains (160 amino acids), andshows no homology to any of the cadherin cytoplasmic domains or tocytoplasmic domains of other proteins to which it has been compared in acurrent sequence data base.

[0126] To confirm that the sequenced clone encoded full-length BT-R₁protein, total mRNA was prepared from midguts of M. sexta subjected toNorthern blot by hybridization with the antisense 4.8 kb SacI fragmentof the BT-R₁ cDNA clone. The Northern blot analysis was conducted byhybridizing to the antisense probe at 42° C. and 50% formamide,5×Denhardt's Reagent, 5×SSCP and 50 μg/ml salmon sperm DNA. The filterwas then washed two times with 1×SSC+0.1% SDS and two times with0.15×SSC+0.1% SDS at 42° C. Each wash was roughly 20 minutes. The filterwas then exposed to X-ray film for 24 hours. The 4.8 kb probe hybridizedto a single 5.6 kb band.

[0127] The BT-R₁ clone was translated using rabbit reticulolysate andthe resulting translated products were immunoprecipitated with antiseraraised against native protein encoded by BT-R₁. For the in vitrotranslation, pBluescript plasmid containing BT-R₁ cDNA was linearizedand transcribed with T₃ polymerase (Pharmacia). The translation wasconducted according to manufacturer's instructions with nuclease-treatedrabbit reticulolysate (Life Technologies, Inc.). After one hour ofincubation at 30° C., the reaction mixture was combined with an equalvolume of SDS buffer or lysed with 50 mM Tris buffer containing 1% NP40and 250 mM NaCl (pH 8.0) for immunoprecipitation. Preimmune serum wasused as a control. Translation and immunoprecipitation products wereelectrophoresed on a 7.5% SDS-polyacrylamide gel fixed, treated withEnhance (Dupont NEN), dried and exposed to X-ray film for 12 hours.

[0128] Two protein bands of approximately 172 kD and 150 kD asdetermined by SDS-PAGE were obtained; it is postulated that the 150 kDtranslation product was due to initiation of translation from aninternal methionine at amino acid 242. This is consistent with theobservations of Kozak, M. Mol Cell Biol (1989) 9:5073.

[0129] Thus, both results confirm that a full-length clone was obtained.

EXAMPLE 3 Recombinant Production and Characteristics of the BT-R₁Protein

[0130] The BT-R₁ cDNA clone was subcloned into the mammalian expressionvector pcDNA3 (Invitrogen) and the construct transfected into COS-7cells. Membranes isolated from the COS-7 transfectants were solubilized,electrophoresed and ligand blotted with ¹²⁵I-CryIAb toxin. The cellswere harvested 60 hours after transfection, washed withphosphate-buffered saline and lysed by freezing in liquid nitrogen. Cellmembranes were prepared by differential centrifugation as described byElshourbagy, N. A. et al. J Biol Chem (1993) 266:3873. Control cellswere COS-7 cells transfected with pcDNA3.

[0131] The cell membranes (10 μg) were separated on 7.5% SDS-PAGEblotted to a nylon membrane and blocked with Tris-buffered salinecontaining 5% nonfat dry milk powder, 5% glycerol and 1% Tween-20. Thenylon membrane was then incubated with ¹²⁵I-CryIAb toxin (2×10⁵ cpm/ml)for two hours with blocking buffer, dried and exposed to X-ray film at−70° C. The labeled toxin bound to a 210±5 kD protein; the 210 kD bandwas observed only in lanes containing membranes prepared from either M.sexta or COS-7 cells transfected with the BT-R₁ cDNA constructcontaining 4810 bp of cDNA comprising the open reading frame.

[0132] The discrepancy between the 210 kD protein expressed and thecalculated 172 kD molecular weight is due to glycosylation of theprotein; in vitro translation of the cDNA clone, as described above,which does not result in glycosylation, does produce the 172 kD protein.To verify this, the COS-7 produced protein was subjected to digestionwith N-glycosidase-F by first denaturing the purified protein by boilingin 1% SDS for 5 minutes followed by addition of NP-40 to a finalconcentration of 1% in the presence of 0.1% SDS, and then incubating thedenatured protein in sodium phosphate buffer, pH 8.5 at 37° C. withN-glycosidase-F for 10 hours. Controls were incubated under the sameconditions without enzyme. Digestion products were separated on a 7.5%SDS-PAGE and stained with Coomassie brilliant blue. This glycosidasetreatment reduced the molecular weight of BT-R₁ protein from 210 to 190kD; this indicates N-glycosylation at some of the 16 consensusN-glycosylation sites in the protein. Treatment of BT-R₁ withO-glycosidase and neuraminidase did not alter the mobility of theprotein.

[0133] In addition, embryonic 293 cells were transfected with the BT-R₁cDNA clone in pcDNA3 and incubated with the labeled toxin (0.32 nM) inthe presence of increasing concentrations (0 to 10⁻⁶ M) of unlabeledtoxin. Nonspecific binding was measured as bound radioactivity in thepresence of 1 TM unlabeled toxin. A value for the dissociation constant(Kd) of 1015 pM was determined by Scatchard analysis; this isapproximately the same value that was obtained for the natural receptoras described by Vadlamudi, R. K. et al. J Biol Chem (1993) (supra).

EXAMPLE 4 Physiological Effect of BT Toxin on Modified Embryonic 293Cells

[0134] Both unmodified embryonic 293 cells, and 293 cells which havebeen modified to produce the BT-R₁ receptor as described in Example 3,when cultured in vitro form adherent star-shaped clusters. When BT toxin(200 nM) is added to serum-free medium, the clusters round up andrelease from the plastic surfaces of the culture dish. This effect isalso observed under known conditions of cytotoxicity for 293 cells. Theforegoing effect is observed only when the cells are cultured inserum-free medium since the toxin binds to serum and would thus beineffective under conditions where serum is present.

[0135] However, in the presence of anti-receptor antisera, this effectof BT toxin is blocked. Also, when serum is added back to a culture ofmodified E293 cells which has been treated in serum-free conditions withthe toxin, the cells revert to their normal star-shaped adherent clustershapes. This indicates that the effect of the toxin is reversible.

EXAMPLE 5 Identification of a Fragment of BT-R₁ that Binds to a BT Toxin

[0136] To understand some of the properties of BT-R₁, research has beenundertaken to define the location of the BT-R₁/Cry1Ab protein-proteininteraction. The full-length wild-type amino acid sequence of BT-R₁ isprovided in FIG. 1 with a block diagram of a possible cadherin-likestructure for BT-R₁ shown in FIG. 3. In both figures, restriction digestsites from the cDNA are provided relative to the positions at which theywould disrupt the amino acid coding sequence.

[0137] A small fragment lying between the BamHI and SacI restrictionsites of wild-type BT-R₁ was cloned into the vector pCITE (Novagen).This vector contains transcription/translation sequences designed foruse in a rabbit reticulocyte lysate (RRL) system. The clone has beenanalyzed by restriction mapping and mRNA expression (FIG. 4). Lane UPshows the uncut plasmid and lanes NP and XP show restriction digestsusing NsiI and XhoI, respectively. NsiI is used because it has only onerestriction site lying within the Bam-Sac fragment and does not cutanywhere within the pCITE vector. The BSP lane shows the restrictiondigest of the clone using BamHI and SacI. The digest releases the clonedfragment which separates at about 700 base pairs. The RT1 and RT2 lanesshow mRNA transcription from the clone after linearization with XhoI.The mRNA separates at the expected 1350 base pairs.

[0138] Protein for analysis has been prepared from this clone in twoways. First, an RRL translation kit was employed to produce protein fromthe mRNA transcription reaction described above. Second, the plasmid wasadded directly to an RRL based transcription and translation (TNT)coupled kit. Protein production was detected using ³⁵S-methionine as atag (FIG. 5). The LCR lane shows production of luciferase protein frommRNA in an RRL kit and the LCT lane is luciferase protein from a plasmidcontaining the luciferase coding sequence translated in the TNT kit.Both are positive controls to demonstrate that the two translation kitsare operational. The major bands for luciferase translation are observedat 66 kDa. The lanes labeled as RR₁ and RR2 show expression of thepolypeptide sequence of the Bain-Sac fragment of BT-R₁ translated frommRNA in the RRL kit. The lanes TT1 and TT2 are translations from thepCITE plasmid containing the Bam-Sac fragment from the TNT kit. All fourlanes possess a major band at 30 kDa which is the expected size of theBam-Sac fragment with the addition of a coded antibody tag called S-tag.S-tag is part of the multicloning site of pCITE.

[0139] The clone was then tested for its ability to bind theinsecticidal toxin Cry1Ab. Polypeptide translation of the Bam-Sacfragment of BT-R₁ was carried out in duplicate as described above. Theonly change is that the ³⁵S-methionine tag was left out of the reactionmixtures to produce non-radiolabeled proteins. The proteins wereseparated by SDS-PAGE, blotted to nitrocellulose and hybridized with¹²⁵I-labeled Cry1Ab (FIG. 6). BBMV is wild-type BT-R₁ prepared from themidgut brush border membrane vesicles (RBBMV) of M. sexta and, is usedas a positive control. RBK and TBK are RRI, and

[0140] TNT control reactions prepared without mRNA or plasmid present todetermine whether proteins endogenous to either kit bind Cry1Ab. R₁ andRR2 are translations from the RRL kit and TT1 and TT2 are from the TNTkit. A single 30-kDa band appears in each of these lanes. Two are markedby arrows. These bands demonstrate that the Bam-Sac fragment of BT-R₁ iscapable of binding Cry1Ab insecticidal toxin.

[0141] To further understand the nature of this binding site, a set oftruncation mutants of BT-R₁ was prepared through the use of restrictiondigests. The cDNA was digested at specific sites to remove increasinglylarger portions of the C-terminus. The restriction enzymes used wereNsiI, BamHI, NruI, ClaI, XhoI and StuI (FIGS. 1 and 3). The procedureinvolved linearizing the plasmid at each one of these sites andtranscribing up to the truncation. The shortened mRNAs then weretranslated in an RRL kit blotted to nitrocellulose and hybridized with¹²⁵I-labeled Cry1Ab. Translation of the wild-type BT-R₁ from the cDNAshowed binding to a 172-kDa protein band, the expected size of wild-typeBT-R₁. It also shows smaller bands that bind Cry1Ab although the natureof these bands has not been determined. A blank made by preparing an RRLreaction mixture without any mRNA gaves several bands below 66 kDa thatshow some type of binding of Cry1Ab to the reticulocytes. Thespecificity of this binding has not been determined. The truncationmutants created by NsiI, BamHI, NruI, ClaI, XhoI and StuI restrictiondigests did not show any binding to Cry1Ab except in the region wherethe reticulocytes bind Cry1Ab. This data demonstrates that the removalof the last 100 amino acids from wild type BT-R₁ by NsiI restrictionresults in the loss of the ability of BT-R₁ to bind Cry1Ab. Thislocalizes the toxin binding site on the BT-R₁ clone and provides asoluble fragment of the receptor that can be used in toxin and otherbinding studies.

[0142] A clone of a fragment of BT-R₁, called the Bam-Sac fragment, hasbeen prepared. It was prepared using BamHI and SacI restriction digests(FIG. 1) and cloning of the resulting fragment into a vector calledpCITE. The polypeptide sequence was translated and tested for binding tothe insecticidal toxin Cry1Ab (FIG. 8). The Bam-Sac fragment binds toCry1Ab, providing first insight into the location of the Cry1Ab bindingsite within the BT-R₁ sequence. It lies in the last 234 C-terminal aminoacids. This evidence is further supported by a set of truncation mutantsthat has been prepared. Removal of the 100 most C-terminal amino acidsfrom wild type BT-R₁ results in the loss of Cry1Ab binding. TheC-terminal end of BT-R₁ is the location of the Cry1Ab binding site.

EXAMPLE 6 Identification of Homologue of BT-R₁ that Binds to a BT Toxin

[0143] Western blots of tissue extracts prepared from Pink bollworm andEuropean corn borer were prepare and probed with labeled Cry1a (FIG. 7).The results show that homologues of BT-R₁ are present in these twoinsects and can be readily isolated using the methods described herein.

1. A method to identify agents that bind to a BT-toxin receptor, saidmethod comprising the steps of: i) contacting an agent with a BT-toxinbinding receptor selected from the group consisting of a) a cell thathas been altered to contain a nucleic acid molecule that encodes theamino acid sequence of SEQ ID No:2, b) a cell that has been altered tocontain a nucleic acid molecule that encodes a fragment of the aminoacid sequence of SEQ ID No:2 that binds to a BT toxin, c) a cell thathas been altered to contain a nucleic acid molecule encoding a BT-toxinreceptor that hybridizes to the nucleic acid sequence of SEQ ID No:1under high stringency, d) a cell that has been altered to contain anucleic acid molecule that encodes a fragment of a BT-toxin receptorthat hybridizes to the nucleic acid sequence of SEQ ID No:1 under highstringency and that binds to a BT toxin, e) an isolated protein with anamino acid sequence of SEQ ID No:2, f) an isolated fragment of a proteinwith an amino acid sequence of SEQ ID No:2, said fragment containing aBT-toxin binding domain, g) an isolated BT-toxin receptor that isencoded by a nucleic acid molecule that hybridizes to the nucleic acidsequence of SEQ ID No:1 under high stringency, and h) an isolatedfragment of a BT-toxin receptor that is encoded by a nucleic acidmolecule that hybridizes to the nucleic acid sequence of SEQ ID No:1under high stringency, and ii) determining whether said agent binds tosaid BT-toxin receptor.
 2. The method of claim 1, wherein said methodfurther comprises the step of determining whether said agent blocks thebinding of a BT-toxin to said BT-toxin receptor.
 3. The method of claim1, wherein said cell that has been altered is a eukaryotic cell.
 4. Themethod of claim 3, wherein eukaryotic cell is an insect cell.
 5. Amethod to identify agents that block the binding of a BT-toxin to aBT-toxin receptor, said method comprising the steps of: i) contacting anagent, in the presence and absence of a BT-toxin, with a BT-toxinbinding receptor selected from the group consisting of a) a cell thathas been altered to contain a nucleic acid molecule that encodes theamino acid sequence of SEQ ID No:2, b) a cell that has been altered tocontain a nucleic acid molecule that encodes a fragment of the aminoacid sequence of SEQ ID No:2 that binds to a BT toxin, c) a cell thathas been altered to contain a nucleic acid molecule encoding a BT-toxinreceptor that hybridizes to the nucleic acid sequence of SEQ ID No:1under high stringency, d) a cell that has been altered to contain anucleic acid molecule that encodes a fragment of a BT-toxin receptorthat hybridizes to the nucleic acid sequence of SEQ ID No:1 under highstringency and that binds to a BT toxin, e) an isolated protein with anamino acid sequence of SEQ ID No:2, f) an isolated fragment of a proteinwith an amino acid sequence of SEQ ID No:2, said fragment containing aBT-toxin binding domain, g) an isolated BT-toxin receptor that isencoded by a nucleic acid molecule that hybridizes to the nucleic acidsequence of SEQ ID No:1 under high stringency, and h) an isolatedfragment of a BT-toxin receptor that is encoded by a nucleic acidmolecule that hybridizes to the nucleic acid sequence of SEQ ID No:1under high stringency, and ii) determining whether said agent blocks thebinding of said BT-toxin to said BT-toxin receptor.
 6. The method ofclaim 5, wherein said BT-toxin is a member of the BT-cry(1) family oftoxins.
 7. The method of claim 5, wherein said cell that has beenaltered is a eukaryotic cell.
 8. The method of claim 7, whereineukaryotic cell is an insect cell.
 9. An isolated antibody, wherein saidantibody binds to a protein selected from the group consisting of a) aBT-toxin receptor protein with an amino acid sequence of SEQ ID No:2,and b) a BT-toxin receptor protein that is encoded by a nucleic acidmolecule that hybridizes to the nucleic acid sequence of SEQ ID No:1under high stringency, or a fragment of said antibody, wherein saidantibody fragment binds to said BT-toxin.
 10. The antibody of claim 9,wherein said antibody binds to said BT-toxin receptor and blocks thebinding of a BT-toxin to said receptor.
 11. The antibody of claim 10,wherein said antibody binds to an epitope located within the 232c-terminal amino acids of the BT-toxin receptor depicted in SEQ ID No:2.12. An isolated BT-toxin receptor protein selected from the groupconsisting of a) a BT-toxin receptor protein with an amino acid sequenceof SEQ ID No:2, b) a BT-toxin receptor protein that is encoded by anucleic acid molecule that hybridizes to the nucleic acid sequence ofSEQ ID No:1 under high stringency, c) a fragment of a BT-toxin receptorprotein with an amino acid sequence of SEQ ID No:2, said fragment beingable to bind to a BT-toxin, and d) a fragment of a BT-toxin receptorprotein that is encoded by a nucleic acid molecule that hybridizes tothe nucleic acid sequence of SEQ ID No:1 under high stringency, saidfragment being able to bind to a BT-toxin.
 13. A method to produceBT-toxin receptor protein, or a fragment thereof, said method comprisingthe steps of: i) culturing a cell that has been altered to contain anucleic acid molecule that encodes a BT-toxin receptor protein, ofBT-toxin binding fragment thereof, wherein said cell has been altered tocontain a nucleic acid molecule selected from the group consisting of a)a nucleic acid molecule that encodes the amino acid sequence of SEQ IDNo:2, b) a nucleic acid molecule that encodes a fragment of the aminoacid sequence of SEQ ID No:2 that binds to a BT toxin, c) a nucleic acidmolecule encoding a BT-toxin receptor that hybridizes to the nucleicacid sequence of SEQ ID No:1 under high stringency, and d) a nucleicacid molecule that encodes a fragment of a BT-toxin receptor thathybridizes to the nucleic acid sequence of SEQ ID No:1 under highstringency and that binds to a BT toxin, under condition in which saidnucleic acid molecule is expressed and ii) isolating said BT-toxinreceptor protein or fragment.
 14. The method of claim 13, wherein saidcell that has been altered is a eukaryotic cell.
 15. The method of claim14, wherein eukaryotic cell is an insect cell.